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Question 1: 5' A B C D 3' 5' a b c D 3' How might one get the following recombinant from the parental strands above, using the Holliday model for recombination? 5' A b c D 3' If the first parental DNA molecule (ABCD) was delivered to a recipient cel (abcD) via Hfr mating, which proteins would be involved in this recombination? Question #2 RNA pol holoenzyme with ? 28 recognizes promoters upstream of flagelar genes. The consensus -35/-10 sequences for these promoters are TAA (16bp spacer) CGATAT. The promoter for a single gene (no other genes in an operon with it) coding for a known flagelar protein has the following base sequence at the -35/-10 regions: TAC (15bp spacer) GGATAT Is this likely to be an abundant flagelar protein, asuming there are not any other transcription enhancers for its expresion? Imagine the cel needed to expres more of the protein as a regular feature of its growth. Propose two changes that might acomplish this goal. Question #3 MalT is the activator of mal gene expresion in E. coli, which is needed for normal growth on the sugar maltose. You wish to identify a malT mutation that gives constitutive expresion of MalT- dependent promoters, and you have a Mal+ strain with its only copy of the lacZ gene (coding for ?-galactosidase) expresed from a mal promoter. What is the phenotype of this strain before mutagenesis on agar plates + X-gal with and without maltose? (Hint: does MalT activate transcription from a mal promoter-alowing ?-galactosidase synthesis-in the absence of maltose?) What type of mutagenesis might you do (transposon insertion, chemical, uv, mutator strain) to get MalT constitutive mutants (MalT c ). What type of mutation (deletion, frameshift, transposon insertion, amber, misense) in malT would you expect to give a MalT c phenotype? Why? After your mutagenesis, how would you plate your mutagenized cels to isolate MalT c mutants? (Hint: medium with or without maltose? With or without X-gal?) Question #4 You isolate a strain of E. coli that contains a plasmid with resistance to ampicilin (100 µg/ml) and wish to study it. You find that the plasmid can be mobilized to a recipient cel by the F plasmid and caries the lacZ gene (medium blue on nutrient agar plate with X-gal). You place this plasmid in a ?lacZ strain after mutagenizing the plasmid by uv and find these diferent mutants. Clas I: mobilizable, medium blue on X-gal, ampicilin-sensitive (Amp S ) Clas I: mobilizable, white on X-gal, Amp R (100 µg/ml) Clas II: mobilizable, dark blue on X-gal, Amp R (500 µg/ml) Clas IV: cannot be mobilizable, medium blue on X-gal, Amp R Clas V: mobilizable, dark blue on X-gal, Amp R (100 µg/ml, but not 500 µg/ml) Propose genetic explanations for each of these mutant phenotypes. Question 5 In a first culture tube, you mix a culture of lamB+ E. coli with a ?N- mutant phage (ratio: 1 bacterial cel to 1 phage). In a second tube, you mix your E. coli culture with a ? deleted for atP (? ?attP). You spread a dilution of cels from each mixture onto an agar plate, such that you would get 100 colonies of cels (if you had not added phage). What do you se on your two agar plates after overnight incubation? If you se colonies, wil they be lysogens? The mixture of E. coli with ? ?attP (from above) is streaked on a plate across a sample of ?N-. Are you likely to se lysogens forming in the area after the cels have crossed this second phage? If so, which specific prophage(s) would be present? Question 6 You have isolated five independent mutants of E. coli that are auxotrophic for the amino acid histidine (His ? ). You patch the five His ? mutants onto minimal glucose (MinGlu) agar with and without added histidine to look for cross-feding. The results of this experiment are shown in the diagram below. Based on these results, what is the minimum number of genes you have identified in the histidine biosynthesis pathway? Diagram the steps of the pathway, labeling each arow with the number(s) of the specific mutant(s) blocked for that particular step. 1 3 4 5 5 4 4 2 3 2 4 1 3 4 5 5 4 2 3 2 MinGlu + His MinGlu Beth Traxler ProblemSet3

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